PROJECT SUMMARY The prevalence of obesity has reached pandemic levels with nearly 2 billion people worldwide considered overweight and one-third of that obese. Obesity is associated with an increased risk for type II diabetes, heart disease, and certain types of cancer which severely debilitates quality of life and adds considerably to national healthcare budgets. While diet and lifestyle choices are the main contributors, dysregulation of adipogenic or metabolic pathways also predispose individuals towards obesity. Thus, it is essential we understand the underlying mechanisms of adipocyte development and metabolism to help address this global health crisis. Traditionally, we have relied upon knockout or knock-in approaches to study the function of adipogenic genes in a physiological context. While this method has been useful to uncover mechanistic pathways, it remains inefficient as generating transgenic mice is time consuming, challenging, and expensive. Moreover, it limits the scope of gene targets as embryonically lethal genes cannot be explored. To circumvent these issues, we developed a robust, adipose-specific CRISPR/Cas9 model to improve our ability to study adipocyte physiology. CRISPR/Cas9 enables an unprecedented ability to target the genome and introduce permanent, site specific mutations in DNA. Recently, several strategies to model disease in vivo have utilized CRISPR/Cas9 via delivery or transgenic expression of its components in a tissue specific manner. The use of CRISPR/Cas9 to explore gene function in adipose tissue, however, has remained limited. Our approach focuses on an adipose- specific inducible Cas9-mouse model to which single-guide RNAs (sgRNAs) are delivered to target genes of interest. This method is innovative in that it: a.) reduces the time required to generate a transgenic mouse, b.) affords the ability to probe multiple gene targets simultaneously, c.) allows targeting of embryonically lethal genes in adult mice, and d.) controls Cas9 activity to limit off-target effects. We have created a cloning pipeline strategy and efficiency assay to validate sgRNAs in cultured Cas9-preadipocytes and are optimizing sgRNA delivery to Cas9 mice using adeno-associated virus (AAV) and lipid nanoparticles (LNPs). To show proof of concept, we initially targeted leptin, as successful knockout is easily observed by an obese phenotype. Currently, we are expanding this approach to demonstrate its utility to understand the role of cold-inducible RNA binding protein in adipocyte cold adaptation. Ultimately, this approach can transform how we study adipose tissue and uncover novel therapeutic targets for treating obesity and its associated morbidities.